The disclosure is in the field of protein labeling and detection. More specifically, the disclosure relates to the use of molecular tags to identify and track recombinant molecules, such as small proteins, in a subject.
Transforming growth factor-xcex21 (TGF-xcex21) is a growth factor and immunomodulatory cytokine that is secreted from cells and acts through specific binding interactions with a collection of different cell-surface localized receptors. TGF-xcex21 is the prototype for a large family of secreted polypeptides that includes the three mammalian TGF-xcex2 isoforms (TGF-xcex21, TGF-xcex22, and TGF-xcex23), bone morphogenesis proteins (BMPs), activins, and Mxc3xcllerian inhibitory substance (MIS). More distantly related members of this protein family include murine nodal gene products, Drosophila decapentaplegic complex gene products, and Vg1 from Xenopus.
In general, TGF-xcex2 family proteins are homodimers, wherein each functional protein complex includes two identical, associated monomer subunits. The crystal structure of the TGF-xcex21 homodimer is known (Hinck el al., Biochem., 35:8517-8534, 1996; Qian et al., J. Biol. Chem., 271:30656-30662, 1996). TGF-xcex2 is a very compact protein, having four intramolecular disulfide bridges within each subunit, as well as one intermolecular disulfide bridge.
Each monomer of the protein is synthesized as a large (xcx9c55 kDa) precursor molecule with a long (about 278 residue) N-terminal pro-region and a much shorter (112 residue, 12.5 kDa) C-terminal active domain (the mature region). During the maturation process, two precursor molecules associate with each other; the pro-region is important for proper folding of and proper association between the two active domain monomers. The pro-region of each monomer is proteolytically cleaved from the associated active domain; in most instances however, the pro-region remains associated with the mature TGF-xcex2 fragment. The severed pro-region is referred to as the xe2x80x9clatency-associated peptidexe2x80x9d (LAP). LAP is responsible for blocking the correctly folded TGF-xcex2 homodimer so that it does not interact with its receptor. For an excellent discussion of TGF-xcex2 synthesis, see Khalil, Micro. Infect., 1:1255-1263, 1999.
TGF-xcex2s and their receptors are expressed in essentially all tissues, and have been found to be important in many cellular processes. These include cell growth and differentiation, immunosupression, inflammation, and the expression of extracellular matrix proteins. By way of example, in animal models TGF-xcex2 has been shown to attenuate the symptoms associated with various diseases and disorders, including rheumatoid arthritis, multiple sclerosis, wound healing, bronchial asthma, and inflammatory bowel disease, and has been used in the clinical setting to enhance wound healing.
TGF-xcex21 was the first identified member of the TGF-xcex2 family, and has been intensely studied for over 20 years. There are some TGF-xcex21 antibodies available, but their usefulness in a clinical setting is limited at least in part because they often display some degree of cross-reactivity to other TGF-xcex2 family proteins (see, e.g., U.S. Pat. No. 5,571,714). In most experiments, TGF-xcex2 is iodinated with 125I to enable researchers to track the protein. Radioactive iodination is an expensive and hazardous process, and it usually would be inappropriate to use 125I labeled proteins for in vivo experimentation, for instance in clinical trials.
The ability to track the distribution of any exogenously administered, recombinant forms of TGF-xcex2 family proteins has been restricted by the inability to distinguish between the endogenous forms of the protein produced in treated cells or tissues. In addition, available antibodies to these proteins exhibit some degree of cross-reactivity with related family members.
There have been a few reports of TGF-xcex2 fusions in the literature, but the described molecules have been essentially biologically non-functional. In an effort to produce large quantities of easily purified TGF-xcex2 that retained activity, Nimni and co-workers expressed 6x His-tagged TGF-xcex2 fusion proteins in Escherichia coli (Tuan et al., Conn. Tiss. Res., 34:1-9, 1996; Han et al., Prot. Expr. Purif., 11:169-178, 1997). Serious difficulties were encountered in refolding the denatured fusion protein, and full biological activity was not retained using this system. In addition, the Nimni constructs cannot be used to express a tagged TGF-xcex2 in a mammalian host, since the constructs lack a part the TGF-xcex2 pro-protein (the LAP), which is essential for secretion and proper folding of the TGF-xcex2 protein. In an earlier effort, Wakefield et al. (Growth Factors, 5:243-253, 1991) reported attaching an endoplasmic reticulum retention signal to the C-terminus of full-length TGF-xcex21, in an attempt to maintain the protein in the cell (rather than secreting it to the extracellular matrix). This construct had no biological activity.
It is believed that all prior efforts to fuse a TGF-xcex2 family protein to a peptide or protein have resulted in biologically non-functional molecules. Therefore a need still exists for TGF-xcex2 family protein fusions that maintain substantial biological activity.
This disclosure provides functionalized TGF-xcex2 fusion proteins that maintain substantial TGF-xcex2 biological activity. These fusion proteins are achieved by placing a functionalizing peptide between the pro- and active (mature) portions of a TGF-xcex2 protein, or at a relatively non-conserved site within the mature region of a TGF-xcex2 protein.
Encompassed herein are functional TGF-xcex2 family fusion proteins that contain a functionalizing peptide portion for detecting, quantifying or providing a specific additional function to the fusion protein and a mature TGF-xcex2 family protein, both as a monomer and in the form of a dimer (e.g., a homodimer). Also encompassed are nucleic acid molecules encoding such fusion proteins, and conservative substitutions of such molecules. This disclosure also provides methods for making and using the fusion proteins described, as well as kits.
The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.